Deciphering the Structural Organization of Scavenger Receptor Class B Type I and Its Impact on Cholesterol Transport

Atherosclerosis is characterized as an inflammatory disease involving interactions between low- and high-density lipoproteins (LDL and HDL, respectively), components of the immune system (macrophages and T cells), and endothelial and smooth muscle cells of the arterial wall. These interactions can lead to the formation of lesions in the artery wall, which can then cause myocardial infarction or stroke upon rupture. Since the risk for cardiovascular disease is inversely correlated with plasma HDL levels, HDL is considered to be “atheroprotective”. The primary atheroprotective characteristics of HDL can be attributed to its role in reverse cholesterol transport, the body's endogenous mechanism for cholesterol excretion. The last step of reverse cholesterol transport involves the binding of HDL to its receptor, scavenger receptor class B type I (SR-BI), followed by subsequent transfer of HDL-cholesteryl ester (CE) into the liver for disposal. One approach to improving cholesterol flux out of the body is to enhance HDL-CE removal via SR-BI. We hypothesize that efficient HDLcholesterol transport is dependent upon the structural organization of SR-BI at the plasma membrane. To test this hypothesis, we designed a spectrum of experiments that tested the importance of specific regions within the extracellular domain of SR-BI and also probed the native oligomeric status of SR-BI within the plasma membrane.;First, we investigated a stretch of hydrophobic residues within the Nterminal half of the extracellular domain of SR-BI and determined that this region (specifically a span of amino acids from position 67 to 221) was critical for HDL binding, selective uptake of HDL-cholesteryl ester (HDL-CE), efflux of free cholesterol (FC) to HDL, and redistribution of FC within the plasma membrane. These results suggest that this hydrophobic region may interact with the plasma membrane.;Second, by creating single cysteine (Cys, C) and Cys-less SR-BI mutants, we determined that all Cys residues in the extracellular domain participated in intramolecular disulfide bond formation, and that the presence of these bonds was required for HDL binding and selective uptake of HDL-CE; however, all but one of these residues (C323) was dispensable for cholesterol efflux, while none were required for redistribution of cholesterol within the plasma membrane. These results suggest that a specific disulfide bonding pattern is required to maintain SR-BI in a conformation that supports these cholesterol transport functions.;We next exploited CD36, a related protein that can bind HDL but is unable to mediate selective uptake of HDL-CE. We created a panel of SR-BI/CD36 chimeric receptors to further delineate the boundaries of the functional subdomains within the extracellular domain and observed that key sub-domains within the N-terminal half of the extracellular domain, that coincidentally overlap with important hydrophobic regions identified in our earlier study, are responsible for HDL binding and selective uptake of HDL-CE. We also identified a subdomain in the C-terminal half of the extracellular domain that may overlap with putative dimerization motifs.;Finally, we utilized bimolecular fluorescence complementation coupled to fluorescence resonance energy transfer (BiFC-FRET) to investigate the oligomeric organization of SR-BI in live cells in the presence and absence of ligand (i.e. HDL). The novelty of this technique lies in its ability to detect interactions between three proteins of interest. Our BiFC-FRET studies suggested that SR-BI exists as a constitutive oligomer in the absence of HDL ligand; binding of ligand then causes a conformational change within the oligomeric complex.;Determining the mechanisms of the cholesterol transport functions of SRBI is crucial in understanding its protective role against atherosclerosis. As the SR-BI/HDL interaction is critical to lowering plasma cholesterol levels, an improved understanding of the structure-function relationships within this receptor-ligand complex will hopefully lead the development of new therapeutic strategies to combat this devastating disease.